Heterocycles are a set of functional groups commonly found in microbially-produced small molecules, including antibiotics and anticancer agents. Herein, we propose to study the biosynthesis of the thiazolylpeptides, a class of heterocycle-containing antimicrobial compounds. An understanding of the biosynthetic origins of this family of compounds will not only provide the foundation for combinatorial biosynthesis of novel analogues of these molecules, but will aid in the discovery of new classes of heterocycle-containing natural products which may exhibit improved pharmacokinetic properties. The gene clusters responsible for producing the thiazolyl peptides have eluded researchers in the past. We recently made the surprising observation that the thiocillins are derived from ribosomally synthesized short peptides that undergo a high degree of posttranslational modification to produce the biologically active natural products. The specific aims described in this proposal are: (1) To elucidate the order of post-translational modifications during thiocillin biosynthesis in Bacillus cereus. Elucidation of the mechanism of thiocillin biosynthesis will require systematic double crossover knockouts of each hypothetical biosynthetic gene. Synthetic intermediates will be identified in knockout strains through high resolution mass spectrometry (HRMS) and NMR analysis of compounds purified by HPLC. (2) To examine the substrate scope of this system in order to biosynthetically generate new thiopeptide analogs. The structural gene which encodes the peptide backbone of thiocillin will be alanine-scanned to determine positions that can be substituted without disrupting posttranslational modification of the peptide. (3) To discover new members of this family of natural products as well as novel classes of heterocycle-containing natural products through bioinformatic analysis of microbial genomes. We used the information garnered through identification of the thiocillin gene cluster to discover a gene clusters of related compounds in other Bacillus strains. We will identify the products of these novel gene clusters through systematic comparison of extracts from bacterial strains which contain the same gene cluster. We will solve the structure of the new molecule using 2D-NMR and HRMS. PUBLIC HEALTH RELEVANCE: Bacterial resistance to antimicrobial agents continues to be a major threat to public health. The thiazolyl peptides are a class of antimicrobial compounds which are effective against a number of Gram-positive bacteria, but exhibit poor pharmacokinetic properties. An understanding of thiazolyl peptide biosynthesis will enable us to identify new biologically active molecules, and generate analogues with improved pharmacokinetic properties, and thus strengthen our arsenal of resistance-free antibiotics.